The invention is directed to facilitating usage by polyketide synthase modules of nascent polyketide chains. More specifically, the invention concerns including intermodule and intramnodule linkers in constructions for synthesis of desired polyketides.
Polyketides are a class of compounds synthesized from 2-carbon units through a series of condensations and subsequent modifications. Polyketides occur in many types of organisms, including fungi and mycelial bacteria, in particular, the actinomycetes. Polyketides are biologically active molecules with a wide variety of structures, and the class encompasses numerous compounds with diverse activities. Tetracycline, erythromycin, epothilone, FK-506, FK-520, narbomycin, picromycin, rapamycin, spinocyn, and tylosin are examples of polyketides. Given the difficulty in producing polyketide compounds by traditional chemical methodology, and the typically low production of polyketides in wild-type cells, there has been considerable interest in finding improved or alternate means to produce polyketide compounds.
The biosynthetic diversity of polyketides is generated by repetitive condensations of simple monomers by polyketide synthase (PKS) enzymes that mimic fatty acid synthases. For instance, the deoxyerythronolide-B synthase catalyzes the chain extension of a primer with several methylmalonyl coenzyme A (MeMalCoA) extender units to produce the erythromycin core.
The cloning, analysis, and recombinant DNA technology of genes that encode PKS enzymes allows one to manipulate a known PKS gene cluster either to produce the polyketide synthesized by that PKS at higher levels than occur in nature or in hosts that otherwise do not produce the polyketide. The technology also allows one to produce molecules that are structurally related to, but distinct from, the polyketides produced from known PKS gene clusters. See, e.g., PCT publication Nos. WO 93/13663; 95/08548; 96/40968; 97/02358; 98/27203; and 98/49315; U.S. Pat. Nos. 4,874,748; 5,063,155; 5,098,837; 5,149,639; 5,672,491; 5,712,146; 5,830,750; and 5,843,718; and Fu, et al., 1994, Biochemistry 33: 9321-9326; McDaniel, et al., 1993, Science 262: 1546-550; and Rohr, 1995, Angew. Chem. Int. Ed. Engl. 34(8): 881-888, each of which is incorporated herein by reference.
PKSs catalyze the biosynthesis of polyketides through repeated, decarboxylative Claisen condensations between acylthioester building blocks. The buildinhg blocks used to form complex polyketides are typically acylthioesters, such as acetyl, butyryl, propionyl, malonyl, hydroxymalonyl, methylmalonyl, and ethylmalonyl CoA. Two major types of PKS enzymes are known; these differ in their composition and mode of a synthesis of the polyketide synthesized. These two major types of PKS enzymes are commonly referred to as Type I or xe2x80x9cmodularxe2x80x9d and Type II xe2x80x9citerativexe2x80x9d PKS enzymes.
The present invention concerns modular PKS. In the Type I or modular PKS enzyme group, a set of separate catalytic active sites (each active site is termed a xe2x80x9cdomainxe2x80x9d, and a set thereof is termed a xe2x80x9cmodulexe2x80x9dexists for each cycle of carbon chain elongation and modification in the polyketide synthesis pathway. The typical modular PKS is composed of several large polypeptides, which can be segregated from amino to carboxy terminii into a loading module, multiple extender modules, and a releasing (or thioesterase) domain. The PKS enzyme known as 6-deoxyerythronolide B synthase (DEBS) is a typical Type I PKS. In DEBS, there is a loading module, six extender modules, and a thioesterase (TE) domain. The loading module, six extender modules, and TE of DEBS are present on three separate proteins (designated DEBS-1, DEBS-2, and DEBS-3, with two extender modules per protein). Each of the DEBS polypeptides is encoded by a separate open reading frame (ORF) or gene; these genes are known as eryAI, eryAII, and eryAIII. See FIG. 1. There is considerable interest in the genetic and chemical reprogramming of modular PKSs (see, e.g., Khosla, 1997, Chem. Rev. 97:2577-2590, and Staunton, et al., 1997, Chem. Rev. 2611-2629, each of which is; incorporated herein by reference).
Generally, the loading module is responsible for binding the first building block used to synthesize the polyketide and transferring it to the first extender module. The loading module of DEBS consists of an acyltransferase (AT) domain and an acyl carrier protein (ACP) domain. Another type of loading module utilizes an inactivated KS, an AT, and an ACP. This inactivated KS is in some instances called KSQ, where the superscript letter is the abbreviation for the amino acid, glutamine, that is present instead of the active site cysteine required for ketosynthase activity. In other PKS enzymes, including the FK-520 PKS, the loading module incorporates an unusual starter unit and is composed of a CoA ligase activity domain. In any event, the loading module recognizes a particular acyl-CoA (usually acetyl or propionyl but sometimes butyryl) and transfers it as a thiol ester to the ACP of the loading module.
The AT on each of the extender modules recognizes a particular extender-CoA (malonyl or alpha-substituted malonyl, i.e., methylmalonyl, ethylmalonyl, and hydroxymalonyl) and transfers it to the ACP of that extender module to form a thioester. Each extender module is responsible for accepting a compound from a prior module, binding a building block, attaching the building block to the compound from the prior module, optionally performing one or more additional functions, and transferring the resulting compound to the next module. The transfer into a module is mediated by the KS domain which is upstream of the remaining catalytic domains. The additional functions are performed by enzymes which comprise a ketoreductase (Kg) which reduces the carbonyl group generated from the condensation to an alcohol, a dehydratase (DH) which converts the alcohol to a double bond, and an enoyl reductase (ER) which reduces the doublebond to a single bond. These catalytic domains appear to be immediately adjacent and not separated by any linking sequences. Collectively, they can be called xe2x80x9cbeta-carbonyl modifyingxe2x80x9d domains. Thus, a particular module may contain none of these activities, only KR, or KR+DH, or KR+DH+ER. Thus, the order of domains from the N-terminus of a particular module is KS, AT, beta-carbonyl modifying domains (if present), ACP. The order, Nxe2x86x92C of the beta-carbonyl modifying enzymes is DH ER KR.
Thus, each extender module of a modular PKS contains zero, one, two, or three enzymes that modify the beta-carbon of the growing polyketide chain downstream of the AT catalytic domain. A typical (non-loading) minimal Type I PKS extender module is exemplified by extender module 3 of DEBS, which contains only a KS domain, an AT domain, and an ACP domain. The next extender module, module 4, contains all three 10 beta-carbonyl modifying enzymes. (The beta-carbonyl modifying enzymes effect such modification on the extender unit that has been added by the previous module.)
Once the PKS is primed with acyl- and malonyl-ACPs, the acyl group of the loading module migrates to form a thiol ester (trans-esterification) at the KS of the first extender module; at this stage, extender module one possesses an acyl-KS adjacent to a malonyl (or substituted malonyl) ACP. The acyl group derived from the loading module is then covalently attached to the alpha-carbon of the malonyl group to form a carbon-carbon bond, driven by concomitant decarboxylation, and generating a new acyl-ACP that has a backbone two carbons longer than the loading building block (elongation or extension).
After traversing the final extender module, the polyketide encounters a releasing domain that cleaves the polyketide from the PKS and typically cyclizes the polyketide.
For example, final synthesis of 6-dEB is regulated by a TE domain located at the end of extender module six. In the synthesis of 6dEB, the TE domain catalyzes cyclization of the macrolide ring by formation of an ester linkage. In FK-506, FK-520rapamycin, and similar polyketides, the ester linkage formed by the TE activity is replaced by a linkage formed by incorporation of a pipecolate acid residue. The enzymatic activity that catalyzes this incorporation for the raparnycin enzyme is known as RapP, encoded by the rapP gene. The polyketide can be modified further by tailoring enzymes, these enzymes add carbohydrate groups or methyl groups, or make other modifications, i.e., oxidation or reduction, on the polyketide core molecule. For example, 6-dEB is hydroxylated at C6 and C12 and glycosylated at C3 and C5 in the synthesis of erythromycin A.
In PKS polypeptides, the regions that encode enzymatic activities (domains) are separated by linker or xe2x80x9cscaffoldxe2x80x9d-encoding regions. These scaffold regions encode amino acid sequences that space the domains at the appropriate distances and in the correct order. Thus, the linker regions of a PKS protein collectively can be considered to encode a scaffold into which the various domains (and thus modules) are placed in a particular order and spatial arrangement. Generally, this organization permits PKS catalytic domains of different or identical substrate specificities to be substituted (usually at the DNA level) between PKS enzymes by various available methodologies. Thus, there is considerable flexibility in the design of new PKS enzymes with the result that known polyketides can be produced more effectively, and novel polyketides useful as pharmaceuticals or for otherpurposes can be made.
PCT publication WO 98/49315, the contents of which are incorporated herein by reference, describes an approach for modifying the enzymatic activities included within modules of a PKS by maintaining the scaffolding intact but replacing catalytic domains with different: catalytic domains. U.S. Ser. No. 09/346,860 filed 2 Jul. 1999, now U.S. Pat. No. 6,221,641. and the corresponding PCT publication WO 00/01838, also filed on that date, and incorporated herein by reference describe alternative methods by altering the hypervariable region of the AT domains so as to alter the specificity for an extender unit and alteration of the KS domains to control stereochemistry. The present invention takes advantage of the approach of manipulating modules so that the catalytic activities of an entire module are placed in the appropriate sequence to construct a desired polyketide. The ability to utilize this approach depends on effecting an appropriate means for the module to incorporate a growing polyketide chain, which involves assuring that an appropriate linker region is included. Since the filing of the provisional application from which the present application claims priority, a related paper has been published by Ranganathan, A., et al., Chem. and Biol. (1999) 6:731-741. In this paper, intrapolypeptide linkages are fortuitously supplied to chimeric modules by including the KS region of the native downstream module in a chimera between the corresponding upstream module and the portions downstream of the KS domain in a heterologous module. Alternatively, the downstream module will include the ACP catalytic domain of the native upstream module fused to the remainder of a heterologous module upstream in the chimera.
The invention is directed to an efficient method for constructing an arbitrarily chosen polyketide synthase, and therefore a desired polyketide, by manipulating entire modules of Type I polyketide synthases. The invention enables this approach by providing the modules with the appropriate xe2x80x9clead-inxe2x80x9d or linker sequence to the ketosynthase (KS). Applicants have discovered that the appropriate linker between modules is required upstream of the relevant KS in order to permit the module to accept the nascent polyketide chain, and, in the case of intermolecular transfer, appropriate pairing of N-terrninal and C-terminal regions assures the appropriate transfer. The nature of this linker varies depending on whether the module is covalently linked downstream from another module, or whether it forms the N-termninus of the polypeptide.
Thus, in one aspect, the invention is directed to a method to construct a functional polyketide synthase which method comprises providing each module contained in the desired polyketide synthase with an appropriate intrapolypeptide linker (RAL) when said module is downstream in the same polypeptide from a module derived from a different PKS and with an appropriate interpolypeptide linker (ERL) when the module is derived from a PKS where the module is the N-terminal module of a polypeptide. If the module at the N-terminus of a polypeptide is to accept a nascent polyketide chains from an upstream module, the interpolypeptide linker needs to include the appropriate amino acid sequence at the C-terminus of the module donating the nascent chain.
In describing a xe2x80x9cmodulexe2x80x9d being provided with linker(s) the term xe2x80x9cmodulexe2x80x9d refers to the functional portions extending approximately from the N-terminus 6f the KS catalytic region to the C-terminus of the ACPxe2x80x94i.e., excludes the linker portions otherwise considered a portion of the module.
As further described below, any order of modules of desired specificity can be assured by providing the appropriate linkers either intermolecularly or intramolecularly. Thus, the polyketide synthase can be assembled from individual modules by providing the appropriate linkers to assure that the polyketide chain will be passed in the correct sequence from one module to the next and by assembling these modules either by directly providing the polypeptides containing them or by co-expressing nucleotide sequences and coding them in a host cell.
In other aspects, the invention is directed to materials and compositions useful in carrying out the method, in particular to isolated DNA fragments which contain the appropriate intrapolypeptide and interpolypeptide linkers. The invention also relates to methods to construct functional polyketide synthases from libraries of modules and to polyketides prepared by supplying appropriate substrates to reconstructed polyketide synthases. The polyketides thus prepared can be xe2x80x9ctailoredxe2x80x9d using either isolated enzymes or feeding the polyketides to an organism containing these enzymes to convert them to. anti-infectives or compounds of other activities such as motolides by such post-polyketide modifications as hydroxylation and glycosylation. The ketides or ketolides or their modified forms can also be further derivatized using chemical synthetic methods.